Effect of Physical Structures of Food Matrices on Heat Resistance of Enterococcus faecium NRRL-2356 in Wheat Kernels, Flour and Dough

Nonpathogenic surrogate microorganisms, with a similar or slightly higher thermal resistance of the target pathogens, are usually recommended for validating practical pasteurization processes. The aim of this study was to explore a surrogate microorganism in wheat products by comparing the thermal resistance of three common bacteria in wheat kernels and flour. The most heat-resistant Enterococcus faecium NRRL-2356 rather than Salmonella cocktail and Escherichia coli ATCC 25922 was determined when heating at different temperature–time combinations at a fixed heating rate of 5 °C/min in a heating block system. The most heat-resistant pathogen was selected to investigate the influences of physical structures of food matrices. The results indicated that the heat resistance of E. faecium was influenced by physical structures of food matrices and reduced at wheat kernel structural conditions. The inactivation of E. faecium was better fitted in the Weibull distribution model for wheat dough structural conditions while in first-order kinetics for wheat kernel and flour structural conditions due to the changes of physical structures during heating. A better pasteurization effect could be achieved in wheat kernel structure in this study, which may provide technical support for thermal inactivation of pathogens in wheat-based food processing.


Introduction
Many studies have reported that wheat and its related products have been contaminated with Salmonella [1][2][3][4]. These contaminations threaten public health, although the bacteria are mainly located at surfaces of wheat kernels at very low levels with 0.110 ± 0.448 MPN/g Salmonella cells [4]. A series of risks of an increasing microbial level still exist due to cross-contamination between source materials and the external environment during harvesting, storage, and processing [4][5][6]. In addition, a larger sanitary challenge is faced in wheat-based food due to mixing with meat, egg, nuts, or other microbial carriers [7,8], and consumption of not fully cooked foods [9,10]. In the process of wheat source material to wheat-based food, there are three physical structures, including wheat kernels (graininess), wheat flour (powder) and wheat dough (paste). These structural changes would affect the efficiency of the microbial inactivation during different processing practices. An efficient pasteurization method corresponding to changes in physical structures of wheat source materials is urgently needed.

Sample Preparation and Characteristics
To study the influences of different physical structures of food matrices on the heat resistance of bacteria, wheat samples at graininess, powder and paste structures were prepared. For comparing the microbial heat resistance, a key is to control the a w level of food matrices. However, graininess, powder and paste structures cannot exist at the same a w of food matrices. The influence of different physical structures of food matrices on the heat resistance of bacteria was conducted at two a w levels. According to Syamaladevi et al. [13], wheat flour had a wide a w range between 0.10 to 0.85 corresponding to moisture content (MC) from 7.50 to 19.00% wet basis (w.b.) at 20 • C; thus, wheat kernels at original MC of 11.25% w.b. (a w = 0.66) and its milled flour (particle size: −16 mesh) were selected to study the influence of graininess and powder structures of food matrices on the heat resistance of E. faecium. The prepared wheat kernels and flour were conditioned in closed containers at 4 • C for at least 2 d for equilibrium and then were sterilized by autoclaving at 105 • C for 10 min to achieve a background bacteria level below detection (<1 log CFU/g) without any damage to the materials [23,37,38].
Wheat dough (38.50% w.b., a w = 0.99) was prepared by combining it with sterile water at minimum water addition content for dough formation and gently mixing by hand for 5 min, equilibrating for 15 min in the biosafety hood. The minimum water addition content was determined by preliminary tests. Wheat kernel samples were prepared by added pre-calculated weight of sterile distilled water into presterilized wheat kernels and sealed in sterile bags, equilibrating at 4 • C for 2 d, and were gently hand mixed every 2 h until the final a w was equal to that of dough. The wheat kernels with adjusted moisture levels and dough were selected to study the influence of graininess and paste structures of food matrices on the heat resistance of E. faecium. The background bacteria concentrations of the prepared materials with readjusted moisture levels were determined to be less than 1 log CFU/g by plate count method before the HBS heating experiment.
The MC and a w of samples were determined at 105 • C using the oven method and measured at 25 • C by an Aqua Lab water activity meter (Aqua Lab 4 TE, Decagon Devices, Inc., Pullman, WA, USA), respectively. Densities of wheat kernels, wheat flour and dough in the cell (inner diameter: 20.6 mm, height: 3 mm) of HBS were measured by the liquid displacement method [39] and according to the reference [40], respectively. MC, a w, and densities of wheat kernels, wheat flour and dough are shown in Table 1, and MC and a w showed no significant difference between two physical structures of food matrices (p > 0.05). A maximum a w difference less than 0.015 in materials indicated that the influence of a w could be ignored when comparing the heat resistance of bacteria in food matrices within two physical structures [41].

Preparation and Inoculation of Cell Suspension
Each culture was prepared by subjecting to two consecutive transfers (24 h incubation periods at 37 • C) in 30.0 mL and 300.0 mL Luria-Bertani broth for Salmonella and E. coli, and tryptic soy broth supplemented with 0.6% (w/v) yeast extract for E. faecium, respectively [15,20,33]. Two Salmonella cultures were mixed to form a 2-strain cocktail of Salmonella. The bacterial suspensions were centrifuged at 3500 r/min, and their final concentrations were adjusted to 10 11 -10 12 CFU/mL with 200 µL 8.5% saline for Salmonella cocktail and E. coli [20], and 0.1% peptone water for E. faecium [15] used in subsequent experiments and refrigerated for no more 2 d prior to inoculation in materials.
For comparing the influence of graininess and powder structures of food matrices on the heat resistance of E. faecium, an aliquot of 10 µL bacteria suspension was inoculated in~0.7 g wheat kernels and flour by gently hand mixing for 1 min and then dried for~40 min inside a biosafety hood until the a w of the sample equaled to that of a non-inoculated sample [12,15]. By simulating bacterial liquid distribution with the blue ink, the inoculation uniformity was determined by the color distribution with photos that the deviations of the L* and b* values were less than 8% and 7%, respectively, according to preliminary tests.
To investigate the effect of graininess and paste structures of materials on most heat-resistant bacteria, about 0.9 g wheat kernels and dough were inoculated by the same steps as described above. The drying time was determined by preliminary tests according to the a w of the sample using the water activity meter [2,12,15]. After equilibrating, the inoculated samples were transferred into TDT cells and closed immediately.

Heat Treatment
Heat treatments of E. faecium, Salmonella cocktail, and E. coli when inoculated in materials with three physical structures were conducted in the TDT HBS, which can precisely control the heating rate, set-point temperature and holding time in inactivation kinetic experiments [15,31]. In HBS, six TDT cells (inner diameters: 20.6 mm and height: 3 mm) were designed to hold a small weight capacity to achieve heating uniformity in materials [31]. One TDT cell was filled with uninoculated material to monitor temperature changing, while the other five TDT cells were filled with inoculated samples to perform the heat inactivation of bacterial. The detailed information about TDT HBS can be found in other studies [30,31]. In this study, all heating conditions were conducted at a fixed heating rate of 5 • C/min to simulate the fast-heating rate commonly used in radio frequency pasteurization treatments [33,42]. Various microbial strains may respond to heat differently. The safe treatment protocol in the laboratory should be developed based on the most heat-resistant strain. Since the structure change from flour to dough is caused by MC changing, wheat kernel (a w = 0.66) and flour (a w = 0.65) were selected as the representative model to compare the relative heat-resistance among E. faecium, Salmonella cocktail, and E. coli in food matrices using the HBS regardless of physical structures of food matrices in this experiment. Six temperature and time combinations of 75 • C + 2 min, 80 • C + 0.8 min and 85 • C + 0.2 min in wheat kernels, or 75 • C + 4 min, 80 • C + 1.5 min and 85 • C + 0.5 min in wheat flour were selected to determine the most heat-resistant strain according to preliminary tests.

Thermal Inactivation Kinetics
The most heat-resistant strain was further used to study the influences of different physical structures of food matrices on the heat resistance of bacteria in the HBS. Temperatures of 75, 80 and 85 • C or 60, 62, and 64 • C were chosen to compare the influence of graininess and powder or paste structures of food matrices on the heat resistance of bacteria. Different temperature and time combinations were selected to achieve about 4-log CFU/g reductions during holding time according to preliminary tests.

Enumeration
The TDT cell pulled out when the sample temperature reached the target set-point was defined as a time zero sample, and the remaining cells were removed at five different holding time intervals. All the cells were cooled in 4 • C ice water immediately. To enumerate strain survivors, treated samples after cooling were transferred into 50 mL centrifuge tubes, diluted 1:10 with 8.5% saline for Salmonella cocktail and E. coli [20] and 0.1% peptone water for E. faecium [33], respectively. Appropriate tenfold serial dilutions were spread-plated in duplicate onto Luria-Bertani agar for Salmonella and E. coli [20] and tryptic soy agar supplemented with 0.6% (w/v) yeast extract for E. faecium [33], respectively. The plates were incubated aerobically at 37 • C for 48 h, then the colonies were counted, and the populations were converted to log CFU/g.

Fitting of Kinetic Inactivation
The primary models, first-order kinetic and Weibull distribution models, were used to describe inactivation curves. The first-order kinetic model is described below [15,23]: where N t and N 0 are microbial populations (CFU/g) at isothermal treatment time t and time 0 (min) when subjected to a target temperature; D is the time (min) required to reduce the microbial population by 10-fold (1 log) at a constant temperature ( • C) [43]. The Weibull distribution model is described as follows [20,29]: where δ is the time (min) for the first decimal reduction at a given temperature, and p is the shape parameter, dimensionless, which describes whether the fitting curve is linear (p = 1) or nonlinear (p 1) with a decreasing (p < 1) or increasing (p > 1) inactivation rate with time.
As recommended by references [20,26,44], to eliminate influences of the environmental factors on the shape parameter of the Weibull model, a method with standardization of p-value in a data set was used in this study. It means that a single p-value was estimated from the whole set of survival curves of bacterial, and the δ'-values were re-estimated at a fixed p-value.
To compare the goodness of fit between linear and Weibull models, the Akaike information criterion (AIC) was successfully applied in evaluating various microbial fitting models and can be calculated as follows [45,46]: where RSS is residual sums of squares, n stands for the number of measurements, and p for the number of parameters. The model with the lowest AIC number performs the best.

Microstructure Analysis
To compare the influence of the physical structure of food matrices on wheat samples and the most heat-resistant strain inoculated in wheat samples, wheat kernels (a w = 0.66) and flour were treated in the HBS at 80 • C for 5 min. Wheat kernels (a w = 0.99) and dough were treated in the HBS at 62 • C for 5 min. After heat treatment, all five TDT samples were cooled in ice water (<4 • C) for at least 2 min.
Scanning electron microscope (SEM) (Nano SEM 450, FEI, Hillsboro, OR USA) was used to investigate the microstructure of wheat samples and E. faecium inoculated in wheat samples affected by different physical structures of food matrices before and after heat treatment. Control samples without any treatment and treated samples randomly collected from five mixing TDT samples were prepared for SEM observations. For microstructure analysis of wheat samples, wheat kernel surfaces were cut into thin slices (5 × 5 × 3 mm 3 ) and dried in a drying oven at 40 • C for 24 h. Wheat doughs were cut into thin slices (5 × 5 × 3 mm 3 ) and dried by freeze dryer (FreeZone Plus 2.5 L, LABCONCO, Kansas, MO, USA) overnight. For microstructure analysis of E. faecium, cells inoculated in wheat kernel surfaces, flour and dough were fixed in 2.5% glutaraldehyde at 4 • C overnight. Then, samples were washed twice with phosphate buffer (0.1 M, pH 7.2) for 10 min each time. Samples were dehydrated in a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 100%, and 100% (v/v)) for 10 min per step, and then were further dried by CO 2 . All prepared dried samples were sputter-coated with platinum and analyzed using SEM at an accelerating voltage of 5.0 kV under the high vacuum.

Statistical Analysis
All measurements were conducted in triplicate independent experiments. The parameters of kinetic inactivation models were calculated by the USDA integrated pathogen modeling program (IPMP) [46]. The effect of the different physical structures of food materials was evaluated by one-way analysis of variance and Duncan's multiple comparison tests at a 5% significant level or independent t-tests using the statistical software (17.0 version, SPSS Inc., Chicago, IL, USA). Parameters were re-estimated in the statistical SPSS software. Figure 1 shows the population reductions of E. faecium, Salmonella cocktail, and E. coli when inoculated in wheat kernels (a w = 0.66) and flour (a w = 0.65) at three treatment conditions with a fixed heating rate of 5 • C/min in the HBS. The results showed that heat resistances of all strains were significantly different (p < 0.05) at all temperature-time combinations when inoculated in wheat kernels and flour. The population reductions of E. faecium were significantly lower (p < 0.05) than those of Salmonella cocktail and E. coli whether the bacteria were inoculated in wheat kernels or flour. In addition, E. coli showed a significantly lower heat resistance (p < 0.05) than Salmonella cocktail in wheat kernels and flour, which was contrary to the result in almond [32] and red pepper powder [20]. These different results may be caused by different main components in inoculated foods. Therefore, E. faecium, with the highest heat resistance, was chosen to investigate the influences of food matrices These different results may be caused by different main components in inoculated foods. Therefore, E. faecium, with the highest heat resistance, was chosen to investigate the influences of food matrices with the same components and different physical structures on microbial inactivation kinetics in further experiments.

Influence of Graininess and Powder Structures of Food Matrices on Heat Resistance of E. faecium
The population level in the inoculated wheat kernels (aw = 0.66) and flour (aw = 0.65) were 8.84 ± 0.20 and 9.23 ± 0.34 log CFU/g, respectively (Table 1). Inactivation kinetics of E. faecium inoculated in wheat granules (aw of 0.66) and flour (aw of 0.65) are shown in Figure 2. When heated to a fixed temperature, there were different inactivation degrees of microorganisms (0.24-1.39 log CFU/g), and the reduced population of E. faecium may not affect the thermal inactivation in wheat kernels and flour [11]. Survival data of E. faecium inoculated in wheat kernels and flour fitted well to both primary models with low average root mean square error (RMSE) (<0.5 log CFU/g) and showed a better fit to the linear model for both food matrices with smaller AIC (Table 2).

Influence of Graininess and Powder Structures of Food Matrices on Heat Resistance of E. faecium
The population level in the inoculated wheat kernels (a w = 0.66) and flour (a w = 0.65) were 8.84 ± 0.20 and 9.23 ± 0.34 log CFU/g, respectively (Table 1). Inactivation kinetics of E. faecium inoculated in wheat granules (a w of 0.66) and flour (a w of 0.65) are shown in Figure 2. When heated to a fixed temperature, there were different inactivation degrees of microorganisms (0.24-1.39 log CFU/g), and the reduced population of E. faecium may not affect the thermal inactivation in wheat kernels and flour [11]. Survival data of E. faecium inoculated in wheat kernels and flour fitted well to both primary models with low average root mean square error (RMSE) (<0.5 log CFU/g) and showed a better fit to the linear model for both food matrices with smaller AIC (Table 2). These different results may be caused by different main components in inoculated foods. Therefore, E. faecium, with the highest heat resistance, was chosen to investigate the influences of food matrices with the same components and different physical structures on microbial inactivation kinetics in further experiments.

Influence of Graininess and Powder Structures of Food Matrices on Heat Resistance of E. faecium
The population level in the inoculated wheat kernels (aw = 0.66) and flour (aw = 0.65) were 8.84 ± 0.20 and 9.23 ± 0.34 log CFU/g, respectively (Table 1). Inactivation kinetics of E. faecium inoculated in wheat granules (aw of 0.66) and flour (aw of 0.65) are shown in Figure 2. When heated to a fixed temperature, there were different inactivation degrees of microorganisms (0.24-1.39 log CFU/g), and the reduced population of E. faecium may not affect the thermal inactivation in wheat kernels and flour [11]. Survival data of E. faecium inoculated in wheat kernels and flour fitted well to both primary models with low average root mean square error (RMSE) (<0.5 log CFU/g) and showed a better fit to the linear model for both food matrices with smaller AIC (Table 2).  The D-values of 18.59 ± 1.49, 6.49 ± 0.56, and 2.08 ± 0.21 min for E. faecium inoculated in wheat flour were slightly lower than those of 25.5 ± 1.2, 11.4 ± 1.0, and 2.7 ± 0.2 min in wheat flour (a w = 0.6) at 75, 80 and 85 • C, respectively [18]. The differences in D-values at the same heating temperatures may be due to different a w , heating rate, isothermal treatments, and protocols of bacterial inoculation [15,20,33].
As shown in Table 2, the D-values of E. faecium in wheat flour were higher than those in kernels at temperatures of 75-85 • C. The results showed a higher heat resistance for E. faecium in wheat flour inoculum (powder structure), which was similar to the result of heat resistance of S. enteritidis PT 30 when inoculated in wheat kernels and flour [12]. That is, S. enteritidis PT 30 had a higher D-value of 15.1 ± 0.7 min when inoculated in wheat flour compared to that of 8.9 ± 0.4 min when inoculated in wheat kernels at the same a w of 0.40. The reason for the differences in heat resistance of microorganisms inoculated in the materials with the same composition and a w may be caused by the different physical structures of the inoculated materials [20]. For example, when inoculated in food matrices, the microorganisms were surrounded by flour with smaller particles but loose structure while only inoculated on the surfaces of wheat kernels. The difference in the two physical structures of food matrices contributes to the various heat transfer rate between the food matric and microorganism during the heating process. Therefore, microorganisms on the surface of wheat kernels (graininess structure) were easily inactivated due to the direct heat transfer to microorganisms [20].

Influence of Graininess and Paste Structures of Food Matrices on Heat Resistance of E. faecium
The population levels of the inoculated wheat kernels and dough at a w of 0.99 were 9.37 ± 0.17 and 9.34 ± 0.06 log CFU/g, respectively (Table 1). Inactivation kinetics of E. faecium inoculated in wheat granules (a w of 0.99) and dough (a w of 0.99) are shown in Figure 3. There were different degrees of microorganism reduction (0.12-0.51 log CFU/g) at three fixed temperatures, and the reduced population of E. faecium may not affect the thermal inactivation in wheat kernels and dough [11]. Survival data of E. faecium inoculated in wheat kernels and dough fitted well to both primary models with low average RMSE (<0.5 log CFU/g) and showed a better fit to the linear model for wheat kernels with smaller AIC than the Weibull model for wheat dough (Table 3). The D-values of 18.59 ± 1.49, 6.49 ± 0.56, and 2.08 ± 0.21 min for E. faecium inoculated in wheat flour were slightly lower than those of 25.5 ± 1.2, 11.4 ± 1.0, and 2.7 ± 0.2 min in wheat flour (aw = 0.6) at 75, 80 and 85 °C, respectively [18]. The differences in D-values at the same heating temperatures may be due to different aw, heating rate, isothermal treatments, and protocols of bacterial inoculation [15,20,33].
As shown in Table 2, the D-values of E. faecium in wheat flour were higher than those in kernels at temperatures of 75-85 °C. The results showed a higher heat resistance for E. faecium in wheat flour inoculum (powder structure), which was similar to the result of heat resistance of S. enteritidis PT 30 when inoculated in wheat kernels and flour [12]. That is, S. enteritidis PT 30 had a higher D-value of 15.1 ± 0.7 min when inoculated in wheat flour compared to that of 8.9 ± 0.4 min when inoculated in wheat kernels at the same aw of 0.40. The reason for the differences in heat resistance of microorganisms inoculated in the materials with the same composition and aw may be caused by the different physical structures of the inoculated materials [20]. For example, when inoculated in food matrices, the microorganisms were surrounded by flour with smaller particles but loose structure while only inoculated on the surfaces of wheat kernels. The difference in the two physical structures of food matrices contributes to the various heat transfer rate between the food matric and microorganism during the heating process. Therefore, microorganisms on the surface of wheat kernels (graininess structure) were easily inactivated due to the direct heat transfer to microorganisms [20].

Influence of Graininess and Paste Structures of Food Matrices on Heat Resistance of E. faecium
The population levels of the inoculated wheat kernels and dough at aw of 0.99 were 9.37 ± 0.17 and 9.34 ± 0.06 log CFU/g, respectively (Table 1). Inactivation kinetics of E. faecium inoculated in wheat granules (aw of 0.99) and dough (aw of 0.99) are shown in Figure 3. There were different degrees of microorganism reduction (0.12-0.51 log CFU/g) at three fixed temperatures, and the reduced population of E. faecium may not affect the thermal inactivation in wheat kernels and dough [11]. Survival data of E. faecium inoculated in wheat kernels and dough fitted well to both primary models with low average RMSE (<0.5 log CFU/g) and showed a better fit to the linear model for wheat kernels with smaller AIC than the Weibull model for wheat dough (Table 3).     * All parameter estimates are means ± SD over three independent experimental replications, and RMSE were estimated separately for each data set and determined by IPMP software.
Foods 2020, 9, 1890 10 of 17 As listed in Table 3, the D-values of E. faecium in wheat dough were higher than those in kernels at temperatures of 60-64 • C. The results showed a higher heat resistance for E. faecium in wheat dough (paste structure). The reason for the difference of heat resistances of E. faecium inoculated in materials at the same components and a w may be caused by the porous and irregular physical structures of wheat dough due to its gelatinization [21] compared to the compact structure of wheat kernel during HBS treatments. This structural change may be contributing the microorganisms easier surrounded by starch-protein matrix, resulting in a worse heat transfer with a better adaption of E. faecium to heat stress.

Microstructure Observation by SEM
The control samples of wheat kernels ( Figure 4A0,B0) had uneven surfaces. After heat treatment, some crumples were observed on the surfaces of wheat kernel samples ( Figure 4A1,B1). The elliptical Aand B-type starch granules with various sizes were observed in wheat flour ( Figure 4A2,A3), and starch granules had no obvious changes before and after treatment due to the short time treatment of within 5 min. A starch-protein matrix with a porous structure was observed in control samples of wheat dough ( Figure 4B2), and some starch granules began to swell after heat treatment ( Figure 4B3).
The liquid suspension of E. faecium ( Figure 5A0) and its inoculation in different wheat samples without treatment ( Figure 5A1,A3,B0,B2) had complete and clear profiles. After heat treatment, the E. faecium had different degrees of shrinkages ( Figure 5A4,B3) and damages ( Figure 5A2,B1). Some cells started to shrink in wheat flour ( Figure 5A4), while most cells were slightly shrunk in the wheat dough ( Figure 5B3). While inoculated in wheat kernels, most of the cells were severely shrunk and damaged ( Figure 5A2,B1). Therefore, the changes in cells mentioned above indicated that the E. faecium inactivation was influenced by the physical structure of the food matrices and was more easily inactivated in the graininess structure under the same given heating condition.

Influence of Physical Structures of Food Matrices on Heat Resistance of E. faecium
When food matrices had the same basic compositions, and a w , their particle sizes affected the heat resistance of the E. faecium, which may be caused by their different densities and heat transfer rates [12,20]. In the Weibull model, almost all shape parameters of trials were closed to 1, excluding wheat flour at 85 • C and wheat dough, implying that the inactivation rate of E. faecium was not influenced by temperature changes at the given conditions. A p-value > 1 for wheat flour at 85 • C indicated a shoulder effect, a direct lethal effect of the thermal treatment than the accumulation effect, on E. faecium. P-values < 1 for wheat dough showed a tailing behavior for inactivation of E. faecium in the wheat dough, revealing that the sensitive bacterium inactivated first and the remaining could be the strong ones or adapting to the heat stress. These shape values of models shown in Tables 2 and 3 were influenced by the physical structure of food matrices. Particularly, the changes of physical structures caused by gelatinization of wheat dough ( Figure 4B3) during heating provided more protection for E. faecium due to irregular shape changes. These changes strengthened the heat resistance of E. faecium by adapting to the thermal pressure gradually. Thus, a nonlinear inactivation curve was better fitting with an upward concavity trend (p < 1) for E. faecium in this condition compared to wheat kernels and flour. The physical structure changes caused by gelatinization may affect the heat transfer ability from food matrices to E. faecium and a heat resistance of E. faecium. A comprehensive understanding of the influence of changes of physical structures on microbial thermal resistance requires further investigations [12,21].   To investigate the influence of physical structures of food matrices on the heat resistance of E. faecium, the δ value was re-estimated by the Weibull model with a single p-value. This approach can eliminate the structural correlation between the two parameters of the Weibull models [26]. As shown in Figure 6, the a w , temperature and physical structures of food matrices had no great influence on the p-value with an R 2 of 0.264. This observation is in agreement with the result of Couvert [20]. According to references [20,44,49], Weibull models with a fixed shape parameter can be studied to compare the heat resistance of bacterial by re-estimated δ'-value despite the effect of environmental factors. In this study, re-estimated δ'-values by a constant p-value (p = 0.96) from whole Weibull curves were compared to study the single effect of physical structures of food matrices on heat resistances of E. faecium ( Table 1). The result showed that the average inoculation concentrations of E. faecium had no significant difference (p > 0.05) when the physical structures of food matrices varied, but these structural changes had a significant impact on the heat resistance of E. faecium (p < 0.05). These results also suggest that cells attach concentrations in food matrices were similar at any physical structures, and a less heat resistance of E. faecium was observed in wheat kernels compared to wheat flour or dough at any given temperatures. heterogeneous compositions [5,50]. However, the above speculation needs to be verified by detecting the changes of aw with temperature changing in different attachment locations. An effective pasteurization method could be considered in terms of the specific physical structure of the material. The result obtained in this study indicated that a better thermal inactivation effect could be achieved in wheat kernel structure comparing with wheat flour and dough. Moisture tempering, a process of separating the bran and flour easily by adjusting the MC of wheat [5], is one of the processes during wheat to wheat-based food processing. The relatively high MC (15.0-16.0%) of wheat kernels in the tempering process could be beneficial to the effective inactivation of microorganisms [51,52]. Therefore, a pasteurization process after moistening could be suggested in industrial applications to control the target pathogen in wheat-based food for achieving acceptable food safety and product quality [32,52].

Conclusions
This study demonstrated that E. faecium NRRL-2356 could be used as a surrogate for Salmonella cocktail in wheat kernels and its derivatives due to its most heat-resistant characteristics. The heat resistance of E. faecium was influenced by the physical structures of food matrices. E. faecium showed less heat-resistant behavior in wheat kernel structural conditions. The results also suggested that the moistening stage for wheat kernel structure may be suitable for pasteurization to ensure the hygiene Figure 6. The shape parameter p and associated 95% confidence interval as a function of heating temperature for E. faecium in food matrices with different physical structures.
The above results showed that the changes of physical structures in food matrices had an impact on the heat resistance of the microorganism. With changing physical structures of food matrices, the microorganisms made different response changes under the heat stress. It seems that a more complex survival environment (starch or starch-protein matrix, as shown in Figure 4A3,B2) resulted in a better adaption of microorganisms to heat stress. These adaption differences may be caused by a worse heat transfer between food matrices and microorganisms [20]. Some other studies have pointed out that different compositions of food matrices affect the heat resistance of inoculated microorganisms due to their a w changing at high temperatures [13,14,19]. For example, S. enteritidis PT 30 had a lower D 80 • C -values of 6.9 min in wheat flour corresponding to an increase a w from 0.45 to 0.80 while higher D 80 • C -values of 17.0 min in peanut butter corresponding to a decrease a w from 0.45 to 0.04, respectively. Thus, the different heat resistances of E. faecium inoculated in materials with different physical structures may be attributed by various compositions in different attachment locations with outer grain layers (when inoculated on kernel surfaces) and mixtures with whole wheat kernels (when inoculated in wheat flour or dough) since wheat kernels are made of heterogeneous compositions [5,50]. However, the above speculation needs to be verified by detecting the changes of a w with temperature changing in different attachment locations.
An effective pasteurization method could be considered in terms of the specific physical structure of the material. The result obtained in this study indicated that a better thermal inactivation effect could be achieved in wheat kernel structure comparing with wheat flour and dough. Moisture tempering, a process of separating the bran and flour easily by adjusting the MC of wheat [5], is one of the processes during wheat to wheat-based food processing. The relatively high MC (15.0-16.0%) of wheat kernels in the tempering process could be beneficial to the effective inactivation of microorganisms [51,52]. Therefore, a pasteurization process after moistening could be suggested in industrial applications to control the target pathogen in wheat-based food for achieving acceptable food safety and product quality [32,52].

Conclusions
This study demonstrated that E. faecium NRRL-2356 could be used as a surrogate for Salmonella cocktail in wheat kernels and its derivatives due to its most heat-resistant characteristics. The heat resistance of E. faecium was influenced by the physical structures of food matrices. E. faecium showed less heat-resistant behavior in wheat kernel structural conditions. The results also suggested that the moistening stage for wheat kernel structure may be suitable for pasteurization to ensure the hygiene and safety of wheat kernels.
Author Contributions: B.L. conducted experiments and developed the predicted model for analyzing data and wrote the first version of the manuscript; Y.Z. provided the microbial strains and evaluated the microbial populations; L.Z. helped to analyze data; R.X. helped to develop the models; X.G. helped to prepare the samples; X.K. assisted in conducting the experiments with operating the HBS; S.W. was the PI of the project, guided the experimental design and revised manuscript. All authors have read and agreed to the published version of the manuscript.